M.T. Criado

In vitro induction of memory-driven responses against Neisseria meningitidis by priming with Neisseria lactamica.

Natural immunity against Neisseria meningitidis is acquired during childhood and youth through successive colonizations by commensal Neisseria, carrier N. meningitidis, and other bacterial genera sharing cross-reactive antigens with the meningococci. We have analyzed in mice the ability of Neisseria lactamica strains to induce immunological memory so that, upon a later contact with N. meningitidis, quickly raise protective responses against antigens that show cross-reactivity with meningococcal surface proteins. Sera obtained from mice immunized with N. lactamica and boosted with N. meningitidis were able to kill meningococci, with bactericidal activities variable depending on the immunizing strains used in the assays. Different mixtures of those sera resulted in higher killing activities, which agrees with the idea that successive colonizations by N. lactamica enhance the anti-meningococcal response. The existence of such outer membrane cross-reactive antigens has to be kept in mind when using outer membrane vesicle (OMV)-based anti-meningococcal vaccines because their use can affect colonization by N. lactamica and other species, hampering the natural mechanisms of acquisition of immunity to the meningococci, and leaving its ecological niche free for colonization by undesirable microorganisms.

Cooperation between the components of the meningococcal transferrin receptor, TbpA and TbpB, in the uptake of transferrin iron by the 37-kDa ferric-binding protein (FbpA).

Meningococcal TbpAB complexes TbpA, TbpB and FbpA were purified and used to study their role in the uptake of iron from transferrin to FbpA. Purification was achieved by affinity chromatography techniques, yielding homogeneous, non-denatured and functional material. TbpA could not be separated from TbpB and had to be purified from a TbpB-defective mutant strain. FbpA was able to bind iron from transferrin only when TbpAB complexes, TbpA and/or TbpB, were also present during the interaction. The highest uptake efficiences were obtained with TbpAB complexes or TbpA/TbpB mixtures. We conclude that the TbpA and TbpB molecules form true functional transferrin receptors, that FbpA is able to take iron directly from transferrin when in the presence of the components of the receptor, and that both Tbps are necessary for an optimal operation of the uptake system.

Analysis of TbpA and TbpB functionality in defective mutants of Neisseria meningitidis

Iron uptake analysis suggested that the Neisseria meningitidis transferrin (Tf) binding proteins, TbpA and TbpB, form only one type of receptor complex. Mutants defective in the synthesis of either TbpA or TbpB, but not defective in both proteins, can bind Tf, suggesting that both proteins are surface exposed and function in Tf binding. Also, iron uptake from Tf into the meningococci did not require the presence of both Tbps. The TbpB-defective mutant incorporated c. 37% of the iron taken up by the wild-type strain, but this was insufficient for bacterial growth. The TbpA-defective mutant incorporated c. 50% of the iron taken up by the wild-type strain and was able to grow with Tf as the only iron source. Mouse antibodies specific for TbpA were able to block c. 70% of the iron uptake from Tf in the wild-type strain, whereas they blocked only 22% of iron uptake in the TbpB-defective mutant and did not block uptake in the TbpA-defective strain. These results emphasise that TbpA should be considered in future vaccine trials in which iron-restricted proteins are to be included in the vaccine formulation.

EVALUATION OF CROSS-REACTIVE ANTIGENS AS DETERMINANTS OF CROSS-BACTERICIDAL ACTIVITY IN PATHOGENIC AND COMMENSAL NEISSERIA

Several antisera raised against outer membane vesicles obtained from invasive and carrier Neisseria meningitidis strains and commensal Neisseria and Moraxella catharralis species were assayed to test cross-bactericidal activity on Neisseria meningitidis strains. Results demonstrate that, despite the wide antigenic cross-reactivity previously shown by Western-blotting for the major outer membrane antigenic proteins of all Neisseria species, complement mediated killing shows very variable patterns that can not be predicted on the basis of antigenic cross-reactivity. Results of antibody tritations on homologous and heterologous strains, isotyping, and bactericidal activity of sera raised against denatured purified outer-membrane vesicle proteins, suggest that the responsibility for most of the bactericidal activity of the sera must be conformational and/or shared epitopes not detectable by Western-blotting.

Effect of adjuvants in the isotypes and bactericidal activity of antibodies against the transferrin-binding proteins of Neisseria meningitidis

Twenty-eight Neisseria meningitidis strains of different serogroups, serotypes, and TbpB isotypes were used to test the effect of five adjuvant formulations on the immune response to the meningococcal transferrin-binding proteins (Tbps) in mice. Levels of anti-Tbps antibodies were relatively low when purified TbpA-TbpB complexes were used for immunization, those obtained with the RAS adjuvant being the highest, and the isotype distribution reveals a prevalence of the non-bactericidal IgG1. Specific anti-Tbps antibody levels were five to 125 times higher immunizing with whole outer membrane vesicles, with bactericidal isotypes prevailing, which suggests that presentation of these antigens in their natural conformation is crucial to elicit a good response. Nevertheless, bactericidal activity did not correlate with these characteristics, confirming that it must be also influenced by other factors, and direct evaluation of the killing ability is necessary to draw conclusions about the efficacy of antigens or adjuvants in vaccine design.

Adherence of psychrotrophic bacteria to dairy equipment surfaces.

Psychrotrophic bacteria isolated from raw milk were tested for their ability to adhere to steel, two types of rubber, and glass, materials employed in the construction of milking equipment. The adherence assays were carried out by exposure of the materials to radioactively labelled bacteria in both a buffering solution (Ringers) and milk. The degree of adherence of Gram-positive bacteria was lower (P less than 0.001) than that of Gram-negative bacteria. Glass was the material least prone to bacterial adherence (P less than 0.001); there were no significant differences between the other three materials. Milk was found to inhibit adhesion significantly (P less than 0.05), this inhibition being more evident with the most adherent bacteria. There was no statistically significant correlation between bacterial surface hydrophobicity and adherence. Our results suggest that intrinsic bacterial adherence cannot be considered a relevant factor in the contamination of milking equipment.

Induction of immune responses by purified outer membrane protein complexes from Neisseria meningitidis

A broad-spectrum vaccine against disease caused by serogroup B of Neisseria meningitidis is still a challenge due to antigenic variability. In the present study outer membrane protein complexes and their components were analysed using non-denaturing 2D electrophoresis and identified using LC/MS-MS and MALDI-TOF. Outer membrane protein complexes were purified from both the wild-type strain H44/76 and their knock-out mutants lacking PorA, PorB, RmpM or FetA. The immune responses elicited by the whole outer membrane vesicles (OMV) and the purified complexes were analysed for bactericidal activity, antibody surface binding, antibody-mediated C3b/iC3b deposition, membrane attack complex (MAC) deposition and induction of opsonophagocytosis, both on the homologous and several heterologous strains. The main antigenic complexes found were homomeric, formed by the 60 kDa chaperonin (MSP63) or PorB, or heteromeric, formed by different combinations of PorA, PorB and/or RmpM. The lack of some of these proteins in the OMVs from the knock-out mutants did not affect significantly the immune responses analysed except MAC, which was significantly reduced in the anti-PorA- and anti-PorB- sera, and bactericidal activity, which was absent in the anti-PorA- serum. The sera against purified native complexes showed variable activities against the homologous strain, with greatest responses observed for anti-chaperonin and anti-PorA/PorB/RmpM sera. When tested against heterologous strains, the only anti-complex serum showing consistent responses was that against the 60 kDa chaperonin. The comparison of the responses elicited by the different sera suggests an important role of conformational epitopes, present only in native complexes, in the induction of more effective responses against N. meningitidis.

Blocking of iron uptake by monoclonal antibodies specific for the Neisseria meningitidis transferrin- binding protein 2

The existence of epitopes common to different strains in the Neisseria meningitidis transferrin (Tf)-binding protein 2 (TBP2), combined with the ability of polyclonal anti-TBP2 antibodies to inhibit Tf binding and block iron uptake in this species, led to this study on the effect of anti-TBP1+2 monoclonal antibodies (MAbs) to determine the presence of epitopes inside the Tf-binding region. All MAbs used reacted exclusively with the homologous strain when tested by dot-blots of outer membrane vesicles, with the reaction being specific for TBP2 after SDS-PAGE and electroblotting. In contrast, ELISA and iron-uptake blocking assays were also positive with heterologous strains belonging to Rokbis group II (high mol.wt TBP2). The results confirmed the two group classification proposed by Rokbi and, in contrast to other studies, indicated the existence of epitopes in the Tf-binding region that are common only to strains of Rokbis group II. These epitopes may become denatured after drying for dot-blot assays or after SDS-PAGE and electroblotting.

Different expression and genetic control for the K99 antigen and its associated adhesin

Cultivation of K99+ wild strains and transconjugants in MINCA medium with variable concentrations of glucose, glycerol, alanine, or protein-interfering antibiotics shows that the K99 antigen and its associated adhesin are expressed in different ways. The addition of cAMP to medium containing a K99-repressive concentration of glucose demonstrates that the K99-antigen production is controlled by the cAMP-CRP complex, but this does not occur with the adhesin production. Sub-inhibitory concentrations of protein-interfering antibiotics inhibit the K99-antigen production, but do not correlatively affect the adhesin production. All these experiments suggest that the K99 antigen and the adhesin are different structures whose genes are controlled by different mechanisms.

Influencia de adyuvantes en la capacidad de los anticuerpos anti-Tbps de bloquear la unión de transferrina, la asimilación de hierro y el crecimiento en Neisseria meningitidis

Objetivo Analizar el efecto de cinco adyuvantes en la capacidad de los sueros anti-TbpA/B para bloquear la asimilacion de hierro en Neisseria meningitidis. MATERIAL Y METODOS. Se han ensayado 5 formulaciones de adyuvantes para obtener, en ratones, sueros especificos contra los complejos proteicos de union de transferrina (TbpA/B) purificados de una cepa de N. meningitidis y analizar la influencia del adyuvante en la capacidad de los anticuerpos generados de bloquear la union de transferrina y, en consecuencia, la asimilacion de hierro y el crecimiento de los meningococos. Resultados Todos los sueros fueron capaces de inhibir de manera significativa la union de transferrina, la consiguiente asimilacion de hierro y el crecimiento en la cepa homologa, aunque se observo un claro incremento en los titulos de anticuerpos cuando se emplea RAS como adyuvante (1/3.125 frente a 1/125 con los otros adyuvantes). El efecto de estos sueros en una cepa heterologa, con una TbpB de isotipo diferente al de la cepa inmunizante, fue casi nulo, lo que concuerda con la division descrita para el meningococo en 2 grupos en funcion de la TbpB que posean (isotipos I y II). Conclusiones Contrariamente a lo ya demostrado para otra importante proteina meningococica (FbpA), el uso de diferentes adyuvantes en la inmunizacion de ratones con los complejos TbpA/B no ofrece diferencias en la respuesta inmunitaria producida, excepto en lo relativo al titulo de anticuerpos.